The gene regulatory mechanisms that program cell differentiation from proliferating precursor cells are fundamentally important for development, tissue homeostasis and cancer. Using the dramatic cellular differentiation program of spermatogenesis as a model stem cell lineage, we seek to understand the unique, cell type specific transcription program initiated in the spermatocyte stage that programs cells for terminal differentiation. We discovered that testis-specific TAFs, homologs of components of the general PolII transcription machinery, turn on expression of spermatid differentiation genes, in part by counteracting silencing by the Polycomb machinery in precursor cells. The immediate next challenge is to discover how the testis TAFs act - both directly at promoters to activate robust expression of terminal differentiation genes and in the spermatocyte nucleolus by sequestering the Polycomb transcriptional silencing complex. We will test whether the tTAFs form a testis-specific SAGA-like complex that recruits a histone deubiquitinating enzyme to promoter proximal stalled polymerases, allowing elongation of target transcripts, or instead act in a TFIID-like complex with TBP. We will investigate the role of candidate cofactors that may act with the tTAFs we have identified in a pilot RNAi screen, the mediator subunit Med27 and the orphan nuclear hormone receptor Hr51. Because the tTAFs play such a key role, the mechanisms that program the exquisitely cell type and stage specific expression of the tTAFs themselves are central for understanding the developmental regulatory pathway for cell differentiation. We will investigate the role and mode of action of the BTB-Zn finger transcriptional regulator lola, required for expression of tTAFs in spermatocytes, and its binding partner the Jil1 H3S10 kinase, which is required, like lola, for proper meiotic cell cycle progression and spermatid differentiation. In addition, in a directed genetic screen, we will test candidate transcription regulators expressed at the switch from spermatogonia to spermatocyte to identify additional cofactors that act with the tTAFs and elucidate the regulatory machinery that controls tTAF expression. Our work on the mechanism of action and mode of regulation of the tTAFs in the Drosophila male germ line may provide a paradigm for understanding how development programs cell-type specific terminal differentiation by counteracting repression by the PcG at specific target genes. In addition, understanding the mode of action of the meiotic arrest genes of Drosophila may illuminate mechanisms underlying meiosis I maturation arrest infertility in man. PUBLIC HEALTH RELEVANCE: Short statement of relevance: The goal of this project is to understand the gene regulatory mechanisms that program differentiation of male gametes. In both embryonic and adult stem cell lineages, terminal differentiation genes that are repressed by the Polycomb transcriptional silencing machinery in precursor cells must be robustly activated in a cell type specific and gene- selective manner as cell differentiate. Results of our analysis of the mode of action and regulation of the testis specific TAFs of Drosophila will establish paradigms for how cell type specific alternate forms of core gene regulatory complexes act to control cell differentiation, and how these mechanisms are themselves controlled by the germ line developmental program. Because mutations in the tTAFs cause meiosis I arrest and failure of spermatid differentiation, understanding their regulation and mode of action may illuminate mechanisms underlying the male infertility syndrome meiosis I maturation arrest.